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Nucleobase pairs in DNA match hydrogen-bond donor and
acceptor groups on the nucleobases. However, these can adopt
more than one tautomeric form, and can consequently pair with
nucleobases other than their canonical complements, possibly
a source of natural mutation. These issues are now being revisited
by synthetic biologists increasing the number of replicable
pairs in DNA by exploiting unnatural hydrogen bonding patterns,
where tautomerism can also create mutation. Here, we combine
spectroscopic measurements on methylated analogs of isoguanine
tautomers and tautomeric mixtures with statistical analyses
to a set of isoguanine analogs, the complement of isocytosine, the
5th and 6th "letters" in DNA.

One frontier in synthetic biology seeks to move artificially
expanded genetic information systems (AEGIS) into natural living cells and to
arrange the metabolism of those cells to allow them to replicate plasmids built
from these unnatural genetic systems. In addition to requiring polymerases that
replicate AEGIS oligonucleotides, such cells require metabolic pathways that
biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those
polymerases. Such pathways generally require nucleoside and nucleotide kinases
to phosphorylate AEGIS nucleosides and nucleotides on the path to these
triphosphates. Thus, constructing such pathways focuses on engineering natural
nucleoside and nucleotide kinases, which often do not accept the unnatural
AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the
enzyme engineer to follow the kinase reaction, assays that are easily confused by
ATPase and other spurious activities that might arise through "site-directed
damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural
and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring
the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-
(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-
4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro
and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ~92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ~86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is
substantially beyond current theoretical and technological capabilities. In pursuit of this
goal, scientists are forced across uncharted territory, where they must answer unscripted
questions and solve unscripted problems, creating new theories and new technologies in
ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery
and paradigm changes in ways that analysis cannot. Described here are the products
that have arisen so far through the pursuit of one grand challenge in synthetic biology:
Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using
genetic and catalytic biopolymers different from those that have been delivered to us by
natural history on Earth. The outcomes in technology include new diagnostic tools that have
helped personalize the care of hundreds of thousands of patients worldwide. In science, the
effort has generated a fundamentally different view of DNA, RNA, and how they work.

Nucleoside triphosphates having a 3'-ONH(2) blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH(2) group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH(2) blocking group in "next generation sequencing."

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. the ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).

5'-O-4,4'-Dimethoxytrityl-2'-deoxynucleosides have been synthesized in high yield by the reaction of 2'-deoxynucleosides with 4, 4'-dimethoxytrityl chloride in acetonitrile using poly (4-vinylpyridine)-costyrene (styrene 10%).

A new chelating ion-exchange resin with a spacer CH2-NH-C6H4- based on a microreticular chloromethylated styrene-divinylbenzene copolymer containing alpha-nitroso-beta-naphthol as a functional group has been synthesized. The sorption characteristics for manganese(II), iron(III), cobalt(II), nickel(II), copper(II), and zinc(II) have been investigated over the pH range 1.0-7.0. The resin is highly stable in acidic and alkaline medium. Iron(III) and cobalt(II); copper(II) and iron(III) are separated very effectively in a column operation by stepwise elution. (C) 1998 Elsevier Science B.V. All rights reserved.